Stirring method, cell culture method, stirring apparatus, and cell culture apparatus

ABSTRACT

A stirring method includes measuring, with respect to cells which adhere to an inner surface of a culture vessel to be cultured, an ease of peeling between the culture vessel and the cell and an ease of peeling between the mutual cells, and performing a stirring process on the cells in the culture vessel by determining a process content of the stirring process with respect to the cells in the culture vessel, based on the ease of peeling between the culture vessel and the cell and the ease of peeling between the mutual cells.

CROSS REFERENCE TO RELATED APPLICATION

This application is a continuation application of InternationalApplication No. PCT/JP2010/005414, filed on Sep. 2, 2010, designatingthe U.S., in which the International Application claims a priority dateof Sep. 3, 2009, based on prior filed Japanese Patent Application No.2009-203745, the entire contents of which are incorporated herein byreference.

BACKGROUND

1. Field

The present application relates to a stirring method, a cell culturemethod, a stirring apparatus and a cell culture apparatus used at a timeof culturing cells.

2. Description of the Related Art

When culturing cells, a work of replacing a medium inside of a culturevessel and a work of seeding cells to be cultured in a new medium arerequired. Such works are often carried out by hand by a skilled worker,by taking complication of the works and suppression of occurrence ofcontamination and the like into consideration. Recently, there has beenconducted a culture using stem cells for regenerative medicine, and whensuch culture of cells is conducted by hand, efficiency regarding theculture of cells is poor. In recent years, there has been provided acell culture apparatus which realizes an improvement in efficiencyassociated with a culture of cells by automatically executing theaforementioned works (refer to Patent Document 1: Japanese UnexaminedPatent Application Publication No. 2004-016194).

It has been clear that, when culturing cells by using the cell cultureapparatus as described above, an adhesive strength between a cell and aninner surface (referred to as a surface for adhesion, hereinafter) suchas a bottom surface of a culture vessel to which the cell is adhered,and a relation between the adhesive strength between the cell and thesurface for adhesion and an adhesive strength between respective cellsexert large influence on the culture of cells. For this reason,depending on a combination of a cell to be cultured and a culture vesselused at the time of performing the culture, a density variation of cellsproliferated in a process of performing the culture occurs. Theoccurrence of variation as above becomes a main cause of preventing thecells to be cultured from being efficiently proliferated.

The present application has a proposition to provide a stirring method,a cell culture method, a stirring apparatus and a cell culture apparatuscapable of efficiently culturing cells to be cultured, by preventing adensity variation of cells that occurs in a process of performing theculture.

SUMMARY

A stirring method includes measuring, with respect to cells which adhereto an inner surface of a culture vessel to be cultured, an ease ofpeeling between the culture vessel and the cell and an ease of peelingbetween the mutual cells, and performing a stirring process on the cellsin the culture vessel by determining a process content of the stirringprocess with respect to the cells in the culture vessel, based on theease of peeling between the culture vessel and the cell and an ease ofpeeling between the mutual cells.

Further, a cell culture method includes seeding the cells in the culturevessel, and stirring the cells cultured in the culture vessel, by usingthe stirring method.

Further, the measuring measures the ease of peeling between the culturevessel and the cell and the ease of peeling between the mutual cellsincludes measuring the ease of peeling between the culture vessel andthe cell by transporting the culture vessel at a predeterminedacceleration and stopping the culture vessel, when the cells are seededin the culture vessel and the cells are fixed to the culture vessel, andmeasuring the ease of peeling between the mutual cells by transportingthe culture vessel at a predetermined acceleration and stopping theculture vessel after the cells are cultured in the culture vessel.

Further, a stirring apparatus includes a stirring unit executing astirring process on cells which adhere to an inner surface of a culturevessel to be cultured, a stirring process content determining unitdetermining a process content of the stirring process in the stirringunit, in accordance with a magnitude relation between an ease of peelingbetween the cell and the inner surface of the culture vessel and an easeof peeling between the mutual cells, and a control unitdrive-controlling the stirring unit based on the process content of thestirring process determined by the stirring process content determiningunit.

Further, there is provided an input unit inputting a type of the culturevessel and a type of the cell, and the stirring process contentdetermining unit estimates the magnitude relation based on the type ofthe culture vessel and the type of the cell input by the input unit, anddetermines the content of the stirring process in the stirring unit.

Further, there is provided a storage unit storing stirring processinformation to which the process content of the stirring process iscorresponded, in accordance with the type of the culture vessel and thetype of the cell, and the stirring process content determining unitdetermines the process content of the stirring process in the stirringunit by refereeing to the stirring process information when the type ofthe culture vessel and the type of the cell are input by the input unit.

Further, it is preferable that there are provided a culture vesseltransporting unit giving an acceleration to the culture vessel, anobserving unit observing movements of cells in the culture vessel, andan analyzing unit determining the magnitude relation by analyzing anobservation result of the cells obtained by the observing unit.

Further, it is preferable that the magnitude relation is determined as amagnitude relation between an adhesive strength between the cell and theinner surface of the culture vessel and an adhesive strength between themutual cells.

Further, it is preferable that the magnitude relation is determined as amagnitude relation between a moving distance when the cell adhered tothe culture vessel is peeled off from the culture vessel when applyingthe acceleration and a moving distance of the cell of the mutuallyadhered cells when the mutual cells are peeled off from each other.

Note that it is preferable that the process content of the stirringprocess is formed of the presence/absence of the stirring process, theacceleration given to the culture vessel when executing the stirringprocess and a number of times of giving the acceleration.

Further, a cell culture apparatus includes any one of the stirringapparatuses, and a temperature-controlled room including the stirringapparatus in an inside thereof and maintaining an environment to a stateof culturing the cells.

Further, a stirring apparatus includes a stirring unit executing astirring process with respect to cells which adhere to an inner surfaceof a culture vessel to be cultured, an input unit inputting a type ofthe culture vessel and a type of the cell, a storage unit storing aprocess content of the stirring process determined based on previouslydetermined ease of peeling between the culture vessel and the cell andease of peeling between the mutual cells from a combination of the typeof the culture vessel and the type of the cell input by the input unit,and a control unit drive-controlling the stirring unit based on theprocess content of the stirring process read from the storage unit basedon the type of the culture vessel and the type of the cell.

Further, a cell culture apparatus includes the stirring apparatus, and atemperature-controlled room including the stirring apparatus in aninside thereof and maintaining an environment to a state of culturingthe cells.

According to the present embodiment, it is possible to efficientlyculture cells to be cultured by preventing an occurrence of densityvariation of the cells.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 is a front view illustrating an outline of a cell cultureapparatus.

FIG. 2 is a front view illustrating an outline of the cell cultureapparatus whose outer door is opened.

FIG. 3 is a schematic diagram illustrating a configuration of aninterior of a temperature-controlled room.

FIG. 4 is a schematic diagram illustrating a configuration of a stirringapparatus.

FIG. 5 is a functional block diagram illustrating an electricalconfiguration of the cell culture apparatus.

FIG. 6 is a flow chart illustrating a flow of generation andregistration of an observation schedule.

FIG. 7 is a flow chart illustrating a flow of generation andregistration of an observation schedule using a measurement mode.

FIG. 8A is a diagram illustrating a state of culture vessel immediatelybefore a stage of an observation unit is transported at an accelerationα, and FIG. 8B is a diagram illustrating a state of culture vessel afterthe stage transported at the acceleration α is stopped.

FIG. 9 is a diagram illustrating a movement of suspended cell and amovement of adhered cell point-adhered to a surface for adhesion whenthe stage transported at the acceleration α is stopped.

FIG. 10 is a diagram illustrating a movement of suspended cell and amovement of adhered cell connected to another adhered cell when thestage transported at the acceleration α is stopped.

DETAILED DESCRIPTION OF THE EMBODIMENTS

Hereinafter, a configuration of a cell culture apparatus of the presentembodiment will be described in detail. Note that the cell cultureapparatus described hereinbelow is an example, and the present inventionis not limited to the configuration of the cell culture apparatusdescribed in the present embodiment. As illustrated in FIG. 1 to FIG. 3,a cell culture apparatus 10 includes a first casing 11 in which a samplesuch as a microorganism and a cell is cultured, and a second casing 12housing a control unit 13. In an assembly state of the cell cultureapparatus 10, the first casing 11 is disposed on an upper portion of thesecond casing 12.

The first casing 11 includes a temperature-controlled room 15 in whichan inside thereof is covered by a heat insulating material. Thetemperature-controlled room 15 is connected with an outside by a frontopening 16 formed on a front surface of the first casing 11. The frontopening 16 of the first casing 11 is closed by an inner door 17 and twoouter doors 18 a, 18 b. A packing or the like is provided to aperipheral edge portion of a rear surface side of the inner door 17 andthe outer doors 18 a, 18 b, and when respective doors are closed, theinside of the temperature-controlled room 15 is maintained in anair-tight state. Specifically, when only the inner door 17 is closed, orwhen not only the inner door 17 but also the two outer doors 18 a, 18 bare respectively closed, the inflow of heat into the inside of thetemperature-controlled room 15 from the outside of the cell cultureapparatus 10 and the outflow of heat to the outside of the cell cultureapparatus 10 from the inside of the temperature-controlled room 15 areprevented. Note that the inside of the temperature-controlled room 15 iscontrolled to have previously set environmental conditions (temperature,humidity, carbon dioxide concentration and the like) by a temperatureadjusting device, an atomizing device and the like whose illustration isomitted.

Out of the aforementioned outer doors 18 a, 18 b, the outer door 18 b isprovided with a carry-in door 22. The carry-in door 22 is opened when acarrier 25 housing a culture vessel 31 is carried in a carrierinstallation cradle 26, or when the carrier 25 installed on the carrierinstallation cradle 26 is carried out. When the carry-in door 22 isopened, a small door 24 provided to the inner door 17 via an opening 23is exposed. The inner door 17 and the small door 24 are formed of aglass or a transparent synthetic resin, and it is designed such thateven when the outer doors 18 a, 18 b are opened, environmentalconditions inside of the doors are not changed suddenly. By opening thesmall door 24 provided to the inner door 17, it becomes possible tocarry the carrier 25 housing the culture vessel 31 in the carrierinstallation cradle 26, or to carry out the carrier 25 installed on thecarrier installation cradle 26. Note that the carrier 25 can house threeculture vessels 31 one above the other with a predetermined spacetherebetween, for example.

There are disposed, in the inside of the temperature-controlled room 15,the carrier installation cradle 26, a container 27, a vessel transportmechanism 28, a stirring mechanism 29, an observation unit 30 and thelike. Note that although the illustration is omitted, there aredisposed, in the inside of the temperature-controlled room 15, a mediumreplacement mechanism that performs medium replacement, a seedingmechanism that performs seeding of cells, and the like.

As described above, the carrier 25 is placed on an upper portion of thecarrier installation cradle 26. Although the illustration is omitted, aguide rail is provided to a side surface of the carrier installationcradle 26. The guide rail is fitted into a guide groove provided, on alower surface of the carrier 25, with a space into which the guide railis fitted, thereby regulating a moving direction of the carrier 25 to becarried in or carried out. Note that an extending direction of the guidegroove is orthogonal to a direction in which the culture vessel 31 iscarried in the carrier 25 or a direction in which the culture vessel 31is carried out of the carrier.

The container 27 is disposed on the left side of thetemperature-controlled room 15 when seen from the front of the firstcasing 11. The container 27 has a plurality of shelves in a longitudinaldirection, and the culture vessel 31 is housed in each of the shelves.Note that in the container 27, positions at which the respective culturevessels 31 are housed (referred to as housing positions, hereinafter)are previously set. To each of the housing positions, an identificationnumber for distinguishing between one housing position and anotherhousing position is corresponded, and based on the identificationnumber, an observation schedule of cells to be cultured, image dataobtained at the time of the observation and the like are collectivelymanaged.

As the culture vessel 31 placed in the container 27, a dish, a wellplate, a flask or the like can be cited. In each of the culture vessels31, a sample to be a culture object such as a cell is held together witha medium being a culture solution 33. These culture vessels 31 arehandled in a state of being positioned and fixed to transparenttray-shaped holders 32 when performing the culture of cells and theobservation of cells by the cell culture apparatus 10. Note that as thecell, there can be cited a cell utilized for regenerative medicine suchas a stem cell, for example.

The vessel transport mechanism 28 is disposed substantially on a centerof the temperature-controlled room 15 when seen from the front of thefirst casing 11. The vessel transport mechanism 28 transports theculture vessel 31 housed in the carrier 25 toward the container 27 (orfrom the container 27), or it transports the culture vessel 31 housed inthe container 27 toward any one of the carrier 25, the observation unit30 and the stirring mechanism 29 (or from any one of the carrier 25, theobservation unit 30 and the stirring mechanism 29). Positions ofrespective parts in the vessel transport mechanism 28 are monitored bythe control unit 13 via an encoder or the like.

The stirring mechanism 29 is provided to eliminate a seeding variationwhich occurs at the time of seeding the cells to be cultured in theculture vessel 31, and a density variation of cells which occurs in aprocess of culturing the cells seeded in the culture vessel 31. Notethat a stirring process performed by the stirring mechanism 29corresponds to a process in which at least any one of a vibration in anX direction, a vibration in a Y direction, and a vibration in arotational direction in which a straight line L is set as a center,which will be described later, or a combination of these vibrations isapplied a predetermined number of times to the cells in the mediumtogether with the culture vessel 31. Hereinafter, explanation will bemade by setting such that, among the cells cultured in the culturevessel 31, a cell adhered to a bottom surface (referred to as a surfacefor adhesion, hereinafter) of the culture vessel 31 or a cell connected(adhered) to another cell is called as an adhered cell, and a cell whichis not adhered to the surface for adhesion and is suspended in theculture solution 33 is called as a suspended cell.

As illustrated in FIG. 4, the stirring mechanism 29 includes an X stage35, a Y stage 36, a rotation stage 37, and a base 38. The X stage 35 isdisposed on an uppermost portion of the stirring mechanism 29, and to anupper surface thereof, a plate 39 that performs positioning of theculture vessel 31 is attached. The holder 32 fixing the culture vessel31 is positioned and fixed to an upper surface of the plate 39. The Xstage 35 is moved in an X-axis direction by an X-axis drive mechanism40.

The Y stage 36 is disposed between the X stage 35 and the rotation stage37. The Y stage 36 is moved in a Y-axis direction by a Y-axis drivemechanism 41. When the Y stage 36 moves in the Y-axis direction, the Xstage 35 also moves in the Y-axis direction together with the V stage36.

The rotation stage 37 is disposed between the Y stage 36 and the base38. The rotation stage 37 is rotated around the straight line Lillustrated in FIG. 4 by a rotating mechanism 42. The X stage 35 and theY stage 36 also rotate together with the rotation of the rotation stage37.

Note that in FIG. 4, in order to eliminate complication of drawing, aconfiguration in which the X-axis drive mechanism 40, the Y-axis drivemechanism 41, and the rotating mechanism 42 are disposed on the outsideof the X stage 35, the Y stage 36, the rotation stage 37, and the base38, is employed, but, it is assumed that the mechanisms are actuallydisposed on the inside of the stirring mechanism 29. Each of theaforementioned X-axis drive mechanism 40, Y-axis drive mechanism 41, androtating mechanism 42 is drive-controlled via the later-describedcontrol unit 13. Control contents at the time of driving thesemechanisms (an intensity level at the time of performing stirring, thenumber of times of stirring and the like) are determined based on a typeof cell cultured in the culture vessel 31, a type of culture vessel 31used at the time of performing the culture, and the like.

The stirring mechanism 29 is housed inside of an anti-scattering case45. The anti-scattering case 45 prevents, when the culture solution 33(including cells in the culture solution 33) is scattered from a gapgenerated in the culture vessel 31, the solution from being scattered toanother mechanism in the temperature-controlled room 15. An opening isprovided to a bottom surface of the anti-scattering case 45, and throughthe opening, a connector (illustration is omitted) of the stirringmechanism 29 is inserted. The connector is connected to a connectorprovided to a bottom surface of the temperature-controlled room 15 whenthe stirring mechanism 29 is disposed at a predetermined position in thetemperature-controlled room 15. Note that although the illustration isomitted, a weight sensor whose illustration is omitted is built in thestirring mechanism 29, and the sensor measures a weight of the culturevessel 31 before and after the stirring process performed by thestirring mechanism 29. Note that the measured result is output to thelater-described control unit 13, and when the control unit 13 determinesthat a change in weight of the culture vessel 31 occurs, an alarmdisplay is made on a later-described display unit 65 a. in such a case,the anti-scattering case 45 is detached together with the stirringmechanism 29, and is replaced with new one.

Further, to an upper portion of a side surface facing the vesseltransport mechanism 28 among side surfaces of the anti-scattering case45, an opening 46 is provided. By providing the opening 46, it becomespossible to carry the culture vessel 31 transported by the vesseltransport mechanism 28 in the stirring mechanism 29 disposed inside ofthe anti-scattering case 45, or to carry out the culture vessel 31positioned in the stirring mechanism 29 to the outside of theanti-scattering case 45.

An introduction door 47 is provided to the anti-scattering case 45 sothat it can freely pivot between a position at which the opening 46 isshielded (shielding position) and a position at which the opening isexposed (exposing position). The introduction door 47 is normally keptat the shielding position. When the culture vessel 31 after beingsubjected to the stirring process by the stirring mechanism 29 iscarried out to the outside of the anti-scattering case 45 or when theculture vessel 31 on which the stirring process is to be performed iscarried in the inside of the anti-scattering case 45 to place the vesselon the stirring mechanism 29, the introduction door 47 is pivoted fromthe shielding position to the exposing position by an opening/closingmechanism 48.

To the side surface on the opposite side of the side surface of theanti-scattering case 45 to which the opening 46 is provided, a connector49 is provided. To the connector 49, a connector 52 provided to one endof a tube 51 of a compressor 50 (refer to FIG. 2) is fitted. Thecompressor 50 operates when the culture solution 33 scatters from theculture vessel 31 in a process of performing the stirring process, andsets an air pressure inside of the anti-scattering case 45 to be lowerthan an air pressure inside of the temperature-controlled room 15. Bysetting the air pressure inside of the anti-scattering case 45 to belower than the air pressure inside of the temperature-controlled room15, the culture solution 33 scattered to the inside of theanti-scattering case 45 is prevented from being leaked to the outside ofthe anti-scattering case 45 from the inside of the anti-scattering case45.

A main body of the observation unit 30 is placed inside of the secondcasing 12, and an upper portion of the observation unit 30 is disposedso as to be inserted from a lower part to an inner part of the firstcasing 11. When seen from the front of the first casing 11, theobservation unit 30 is disposed on the right side of thetemperature-controlled room 15. The observation unit is disposed bybeing fitted into an opening on a bottom surface of the first casing 11.Note that although the configuration of the observation unit 30 will beomitted, as the observation unit 30, there can be cited a microscopecapable of performing an observation method of a phase differenceobservation, a fluorescence observation, a differential interferenceobservation or the like.

The observation unit 30 performs the phase difference observation on theculture vessel 31 housed in the carrier 25 immediately after the carrieris installed on the carrier installation cradle 26, or the culturevessel 31 when a fixing time has elapsed after housing the vessel in thecontainer 27, for example, thereby obtaining an entire image of theculture vessel 31 or an image of cells to be cultured. Here, the fixingtime corresponds to a period of time during which the suspended cell inthe culture solution 33 is adhered to the surface for adhesion, forexample, and is 2 hours, for instance. Image data obtained by theobservation unit 30 is stored in a later-described database unit 72.

Next, an electrical configuration of the cell culture apparatus 10 ofthe present embodiment will be described. As illustrated in FIG. 5, thesecond casing 12 houses the main body portion of the observation unit 30described above, and the control unit 13. Further, on a front surface ofthe second casing 12, an operation panel 65 including a monitor 65 a andan input unit 65 b is disposed. Note that as the input unit 65 b, therecan be cited a keyboard, a mouse or the like. To the control unit 13, itis also possible to connect a computer 67 via a communication line 66.

The control unit 13 is connected to the vessel transport mechanism 28,the stirring mechanism 29, the opening/closing mechanism 48, theobservation unit 30, the compressor 50, and the monitor 65 a and theinput unit 65 b of the operation panel 65, respectively. The controlunit 13 comprehensively controls respective parts of the cell cultureapparatus 10 in accordance with a predetermined program. As an example,the control unit 13 automatically determines an observation schedulebased on a type of the cell and a type of the culture vessel 31 used atthe time of performing the culture, input through an operation of theinput unit 65 b of the operation panel 65 or an operation of thecomputer 67. Further, the control unit 13 automatically executes anobservation sequence for the culture vessel 31 based on the determinedobservation schedule. Note that the observation schedule includes notonly a schedule for performing observation of cells cultured in theculture vessel 31 and a schedule for performing medium replacement, butalso a schedule of the stirring process in the stirring mechanism 29 andthe like.

The control unit 13 has a communication section 71, the database section72, a memory 73, and a CPU 74. Note that each of the communicationsection 71, the database section 72, and the memory 73 is connected tothe CPU 74. The communication section 71 executes transmission andreception of data to and from the computer 67 disposed on the outside ofthe cell culture apparatus 10 via a wireless or wired communicationline.

In the database section 72, history information regarding environmentalconditions (temperature, humidity, carbon dioxide concentration and thelike) inside of the temperature-controlled room 15, the image dataobtained by the observation unit 30 and the like are stored. Note thateach of these pieces of data is preferably stored as a management file75 to which an identification number allocated to each of the culturevessels 31 is corresponded.

In the memory 73, schedule data 76 of the observation sequence describedabove, and operation data 77 for calculating an operator at the time ofexecuting the observation sequence are recorded. The schedule data 76 isdata based on the observation schedule. When the stirring process is notincluded in the observation schedule, for example, the schedule data 76is formed of data indicating a start time and a time required forobservation in each observation process, a start time of the mediumreplacement process and a time required for the replacement process, andimage-capturing conditions in the observation unit 30. On the otherhand, the schedule data 76 when the stirring process is included as theobservation schedule is formed of data, in addition to theaforementioned data, based on a schedule regarding the stirring processsuch as data for specifying a start time of the stirring process, a timerequired for the stirring process, stirring conditions (the intensity atthe time of the stirring and the number of times of stirring) of thestirring process in the stirring mechanism 29. The schedule data 76 isstored by being corresponded to the aforementioned identificationnumber, for example. Since the schedule data 76 is only required to bemanaged by being corresponded to the management file 75, it may bestored in the memory 73, separately from the management file 75, or mayalso be stored in the management file 75 stored in the database section72.

Further, in the memory 73, not only the aforementioned schedule data 76and operation data 77 but also first stirring data 78 and secondstirring data 79 are stored. The first stirring data 78 is read when thetype of the cell and the type of the culture vessel 31 are input throughthe operation of the input unit 65 b. The first stirring data 78 is datain which an adhesive strength between a cell and a cell and an adhesivestrength between a cell and a surface for adhesion, the presence/absenceof the stirring process, and an intensity level (intensity of stirring)and the number of times of stirring at the time of the stirring, arerespectively corresponded to a combination of the type of the cell andthe type of the culture vessel 31. Note that as the intensity level atthe time of the stirring, there can be cited an acceleration applied toeach of the stages when the X stage 35 or the Y stage 36 provided to thestirring mechanism 29 are vibrated or when the rotation stage 37 isrotated. Further, the number of times of stirring corresponds to thenumber of times of vibrating each stage, in other words, the number oftimes of applying the acceleration.

The second stirring data 79 is used at the time of executing alater-described measurement mode, The second stirring data 79 is formedof a combination of an adhesive strength generated between a cell and acell when the cells are connected to each other and an adhesive strengthbetween a cell and a culture vessel generated when the cell is adheredto a surface for adhesion, and data to which the presence/absence of thestirring process, the intensity level of the stirring, and the number oftimes of stirring are corresponded, obtained as a result of comparingthese adhesive strengths.

The CPU 74 is a processor that executes various types of operationprocess in the control unit 13. The CPU 74 has functions of an imageanalyzing section 81, an operating section 82, a schedule managementsection 83 and a timer 84. The image analyzing section 81 executes imageanalysis by using the image data obtained by the observation unit 30.The operating section 82 executes operation associated with the imageanalysis. The schedule management section 83 performs a registrationprocess and a changing process of the observation schedule. Further, thetimer 84 is used at the time of managing the observation schedule.

Hereinafter, a flow of process of generating and registering theobservation schedule will be described based on a flow chart in FIG. 6.Note that FIG. 6 illustrates a flow when the type of the cell to becultured and the type of the culture vessel 31 used for the culture arepreviously known.

Step S101 corresponds to a process of inputting the type of the cell andthe type of the culture vessel. A user operates the input unit 65 b toinput the type of the cell and the type of the culture vessel 31. Inthis case, it is also possible to design such that the type of the celland the type of the culture vessel 31 can be directly input through theinput unit 65b, or a list of types of cells and a list of types of theculture vessels 31 are displayed on the monitor 65 a and selection ismade through the operation of the input unit 65 b.

Step S102 corresponds to a process of determining the presence/absenceof the stirring process based on the input type of the cell and type ofthe culture vessel. In step 5101, the type of the cell to be culturedand the type of the culture vessel 31 have been input. The CPU 74 readsthe first stirring data 78 stored in the memory 73, and from datacorresponding to a combination of the type of the cell and the type ofthe culture vessel 31 input through the operation of the input unit 65b, it reads information regarding the presence/absence of the stirringprocess. Based on the read information regarding the presence/absence ofthe stirring process, it is determined whether the stirring process isactually performed or not.

Step S103 is a step of determining whether or not the stirring processis executed. By executing the process in step S102, the informationregarding the presence/absence of the stirring process has been read bythe CPU 74. When the read information regarding the presence/absence ofthe stirring process is information indicating that the stirring processis executed, the CPU 74 sets a result of the determination process inthe step S103 to Yes. In this case, the process proceeds to step S104.On the other hand, when the read information regarding thepresence/absence of the stirring process is information indicating thatthe stirring process is not executed, the CPU 74 sets the result of thedetermination process in the step S103 to No. In this case, the processproceeds to step S106.

Generally, depending on the combination of the cell to be cultured andthe culture vessel 31 in which the cell is cultured, there are variouscases such that the adhesive strength between the cell and the surfacefor adhesion when the cell is adhered to the surface for adhesion of theculture vessel 31 that performs the culture becomes lower or greaterthan the adhesive strength between the cell and the cell when the celland the cell are connected to each other. For example, when the adhesivestrength between the cell and the surface for adhesion when the cell isadhered to the surface for adhesion is lower than the adhesive strengthbetween the cell and the cell when the cells are connected to eachother, the cell to be cultured is easily connected to another cell,resulting in that a portion in which a plurality of cells are connectedto be aggregated (referred to as an aggregated portion, hereinafter) isgenerated. In the aggregated portion, the culture solution 33 does notreach the cell inside of the aggregated portion, so that the cell to becultured dies out. For this reason, when the adhesive strength betweenthe cell and the surface for adhesion when the cell is adhered to thesurface for adhesion is lower than the adhesive strength between thecell and the cell when the cells are connected to each other, it isnecessary to execute the aforementioned stirring process. In such acase, the CPU 74 makes a determination of Yes in the aforementioned stepS103.

On the other hand, when the adhesive strength between the cell and thesurface for adhesion when the cell is adhered to the surface foradhesion is greater than the adhesive strength between the cell and thecell when the cell and the cell are connected to each other, the cellbecomes easily adhered to the culture vessel 31, and becomes difficultto be connected to another cell. Specifically, if the cells arepreviously seeded in a uniform manner, each cell deposits without beingconnected to another cell, and is adhered to the culture vessel 31. Whenthe stirring process is executed in this state, there is a possibilitythat the cell adhered to the culture vessel 31 is peeled off from theculture vessel 31, and is connected to another cell or is died outwithout connecting to another cell, so that in such a case, the stirringprocess is not performed. Specifically, the CPU 74 makes a determinationof No in the step S103,

Step S104 corresponds to a process of determining the intensity ofstirring and the number of times of stirring in the stirring process.The CPU 74 reads the first stirring data stored in the memory 73, andreads the information regarding the intensity level of stirring, and thenumber of times of stirring from the data corresponding to thecombination of the type of the cell and the type of the culture vessel31 input through the operation of the input unit 65 b. Accordingly, theintensity level of stirring and the number of times of stirring in thestirring process to be actually executed with respect to the culturevessel 31 in which the cell is cultured, are determined from the readinformation.

Step S105 corresponds to a process of generating an observation schedulewith the performance of stirring process. The CPU 74 reads the operationdata stored in the memory 73, thereby generating the observationschedule. The CPU 74 makes out a schedule of each process so that theobservation process and the medium replacement process are not executedat the same timing as that of the stirring process, to thereby generatethe observation schedule. At this time, the CPU 74 generates theschedule of the stirring process included in the observation schedule,based on the intensity level of stirring and the number of times ofstirring determined in step S104.

When it is determined that the stirring process is not executed in thedetermination process in step S103, the process proceeds to step S106.Step S106 corresponds to a process of generating an observation schedulewithout the performance of stirring process. As the observation schedulewithout the performance of stirring process, there can be cited aschedule of the aforementioned observation process and mediumreplacement process and the like.

Step S107 corresponds to a registration process of the observationschedule. The CPU 74 makes the observation schedule generated byexecuting step S105 or step S106 correspond to the identificationnumber, and then writes the schedule data 76 on which the observationschedule is based, in the memory 73. Note that based on the scheduledata 76 written in the memory 73, the observation process and the mediumreplacement process of the cells cultured in the culture vessel 31 arerespectively executed, and the stirring process is executed according toneed.

According to the above description, if the combination of the type ofthe culture vessel 31 and the type of the cell to be cultured ispreviously known, and further, the first stirring data 78 correspondingto the combination is stored in the memory 73, it is possible to easilydetermine the presence/absence of the stirring process, and theintensity level and the number of times of stirring in the stirringprocess when performing the stirring process, and further, it ispossible to easily generate the observation schedule in accordance withthe combination of the cell to be cultured and the culture vessel 31 tobe used. By performing the culture of cells based on the observationschedule, it is possible to prevent the occurrence of density variationof cells proliferated in a process of performing the culture, whichenables to efficiently culture the cells.

Some users who perform such culture of cells may culture cells regardingwhich an adhesive force between the culture vessel 31 and the cell to becultured and an adhesive force between the mutual cells to be culturedare not yet registered in the first stirring data 78, and thus there isa case where the determination of the stirring process described abovecannot be performed even when the input is made through the input unit65 b. In order to deal with such a case, for example, the cell cultureapparatus of the present embodiment includes a measurement mode in whichan adhesive strength between a cell and a cell and an adhesive strengthbetween a cell and a surface for adhesion, for example, of a sample ofcells to be actually cultured are determined, and an observationschedule is generated based on these adhesive strengths. Hereinafter,description will be made on a process of generating the observationschedule using the measurement mode, based on a flow chart in FIG. 7.

Step S201 corresponds to a process where the culture vessel 31 iscarried in. The user opens each of the carry-in door 22 of the outerdoor 18 b and the small door 24 of the inner door 17, and sets thecarrier 25 that houses the culture vessel 31 onto the carrierinstallation cradle 26. After the carrier 25 is set onto the carrierinstallation cradle 26, the small door 24 of the inner door 17 and thecarry-in door 22 of the outer door 18 b are respectively closed. Notethat the culture vessel 31 housed in the carrier 25 is preferably theculture vessel 31 that holds the culture solution 33 including cells forgenerating the observation schedule.

Step S202 corresponds to a process of transporting the culture vessel tothe observation unit. When the carrier 25 is set onto the carrierinstallation cradle 26, the carrier 25 is detected by a sensor(illustration is omitted) provided to the carrier installation cradle26. Further, when the carry-in door 22 of the outer door 18 b and thesmall door 24 of the inner door 17 are respectively closed, states ofthe doors are detected by sensors (illustration is omitted) that detectclosed states of the respective doors. Upon receiving detection signalsfrom these sensors, the CPU 74 drives the vessel transport mechanism 28to transport the culture vessel 31 housed in the carrier 25 to theobservation unit 30 from the inside of the carrier 25. Accordingly, theculture vessel 31 is placed on a stage 30 a of the observation unit 30,and positioning of the culture vessel 31 is performed when placing thevessel.

Step S203 corresponds to a process of obtaining images and performingimage analysis. The CPU 74 obtains a plurality of slice images with acertain interval therebetween in an optical axis direction (Z direction)via the observation unit 30. Note that, as is generally known, theseslice images can be obtained by performing confocal observation. The CPU74 generates a three-dimensional image of each cell inside of theculture vessel 31 by using these slice images. Accordingly, a state ofculture of each cell inside of the culture vessel 31 such that each cellinside of the culture vessel 31 is suspended and each cell is adhered tothe culture vessel 31, is identified.

Step S204 corresponds to a process of determining whether or not thereis a cell which is point-adhered to the culture vessel. By the processof step S203, the states of culture of cells inside of the culturevessel 31 have been identified. The CPU 74 determines whether or notthere is a point-adhered cell among the identified respective cells,based on a result of identification in step S203. Hereinafter, a casewhere one cell is point-adhered to the culture vessel 31 will bedescribed, for the sake of convenience. For example, when there is apoint-adhered adhered cell 91, the CPU 74 sets a result of thedetermination process in the step S204 to Yes, and the process proceedsto step S206. When the determination process is performed, the CPU 74writes a position of the point-adhered adhered cell 91 (X-coordinate,Y-coordinate) into the memory 73. At the same time, the CPU 74 specifiesa suspended cell 90, and also writes a position of the suspended cell 90into the memory 73. On the other hand, when there is no point-adheredadhered cell 91, the

CPU 74 sets the result of the process in the step S204 to No. In thiscase, the process proceeds to step S205.

Step S205 corresponds to a process of determining whether or not awaiting time has elapsed. The CPU 74 counts an elapsed time after theexecution of the determination process in step S205, and determineswhether or not the time has reached the waiting time. Note that thewaiting time is a period of time shorter than the fixing time, and is 10minutes, for example. For example, when the CPU 74 determines that thewaiting time has elapsed, the CPU 74 sets a result of the determinationprocess in the step S205 to Yes, and the process proceeds to step S206.On the other hand, when it is determined that a given period of time hasnot elapsed, the CPU 74 sets the result of the determination process inthe step S205 to No. In this case, the process in step S205 isrepeatedly executed.

Step S206 corresponds to a process of measuring the adhesive strengthbetween the cell and the surface for adhesion. First, the CPU 74 drivesthe observation unit 30 to transport the stage 30 a of the observationunit 30 at an acceleration α (refer to FIG. 8A), and stops the stage 30a (refer to FIG. 88). When the stage is stopped and when a given periodof time t has elapsed after stopping the stage, the CPU 74 makes theobservation unit 30 obtain entire images of the culture vessel 31. TheCPU 74 calculates, based on the obtained two entire images, respectivemoving distances of the suspended cell 90 and the adhered cell 91specified by executing step S203. Hereinafter, description will be madeby giving a reference numeral 90 to the suspended cell, a referencenumeral 91 to the adhered cell, and a reference numeral 92 to thesurface for adhesion. After the calculation of these moving distances,an ease of peeling of the adhered cell 91 is determined. Concretely, thepeeled-off cell moves at a certain speed by an acceleration α′ as aresult of subtracting an amount of acceleration consumed for peeling offthe cell from the acceleration given to the adhered cell 91. Therefore,if the moving distance is large, it can be understood that the adhesivestrength is small, and the cell is easily peeled off. Further, on thecontrary, if the moving distance is small, it can be understood that theadhesive strength is large, and the cell is difficult to be peeled off.Conclusively, the adhesive strength between the cell and the surface foradhesion is estimated. Note that the index α′ regarding the accelerationis an acceleration given to the cell point-adhered to the culture vessel31 for peeling off from the culture vessel 31, regardless of the type ofcells. The acceleration α′ is a value which can be previously estimatedthrough experiments, researches, statistics and the like. Further, theindex α′ regarding the acceleration becomes one of indexes indicatingthe adhesive strength between the cell and the culture vessel 31.

As illustrated in FIG. 9, if a mass of each of the suspended cell 90inside of the culture vessel 31 and the adhered cell 91 point-adhered tothe surface for adhesion 92 is set to m, and when the stage 30 a istransported at the acceleration α and then the stage 30 a is stopped, anexternal force F₀ (=mα) due to the acceleration α acts on the suspendedcell 90. Meanwhile, on the point-adhered adhered cell 91, the sameexternal force F₀ as that on the suspended cell 90 acts, and in additionto that, an adhesive force F₁ generated between the cell and the surfacefor adhesion acts in a direction opposite to the direction of theexternal force F₀. For this reason, a force of F₀-F₁(=F₂) acts on thepoint-adhered adhered cell 91, and by the force, the adhered cell 91 ispeeled off from the surface for adhesion 92. The acceleration withrespect to the adhered cell 91 peeled off at this time is set to α′ (α′<a). For example, a moving distance D of the adhered cell 91 and a speedof the adhered cell 91 from when the stage 30 a is stopped to when thegiven period of time t elapses can be roughly determined from theobtained images or coordinate values of respective cells specified fromthe images, so that from these values, it is possible to calculate theindex α′ regarding the acceleration similar to the accelerationgenerated with respect to the peeled-off adhered cell 91, By calculatingthe index α′ regarding the acceleration, the force F₂ that acts on thepeeled-off adhered cell 91 is calculated. By calculating the force F₂,the adhesive force F₁ between the cell and the surface for adhesion isdetermined as the adhesive strength between the cell and the surface foradhesion.

Step S207 corresponds to a process of determining whether or not thefixing time has elapsed. When the step S207 is executed, the CPU 74starts counting of time. When the time counted by the CPU 74 reaches thefixing time, the CPU 74 determines that the fixing time has elapsed. Inthis case, a result of the determination process in the step S207 is setto Yes, and the process proceeds to step S208. On the other hand, whenthe time has not reached the fixing time, the CPU 74 sets the result ofthe determination process in the step S207 to No, and repeatedlyexecutes the process in the step S207 until the time reaches the fixingtime.

Step S208 corresponds to a process of calculating the adhesive strengthbetween the cell and the cell. First, the CPU 74 drives the observationunit 30 to obtain an entire image of the culture vessel 31. The CPU 74performs image analysis using the obtained entire image of the culturevessel 31, and specifies an aggregated portion generated when aplurality of cells are connected.

For specifying the aggregated portion, a binarization process is firstperformed on the obtained entire image of the culture vessel 31. Thebinarization process is a process in which a luminance value exceeding apreviously set threshold value and a luminance value which is equal toor less than the threshold value are divided into two values.Accordingly, it becomes possible to classify a region indicating acontour of the culture vessel 31 and a region of the aggregated portion,and the other regions, for example. Note that the contour of the culturevessel 31 is previously known, so that after the binarization process,the region indicating the contour of the culture vessel 31 is deleted.After these processes, a region with a predetermined size or more isspecified as the aggregated portion. Note that the CPU 74 stores aposition (X-coordinate, Y-coordinate) of the specified aggregatedportion in the memory 73. Hereinafter, description will be made bygiving a reference numeral 95 to the aggregated portion, and a referencenumeral 96 to each of adhered cells that form the aggregated portion 95.

After these processes, the stage 30 a of the observation unit 30 istransported at the acceleration α, and then the stage 30 a which isbeing transported is stopped, in a similar manner to that of the processin step S208. Also at this time, entire images of the culture vessel 31are obtained when the stage 30 a is stopped and when a predeterminedperiod of time t has elapsed after stopping the stage 30 a,respectively, in a similar manner.

By drive-controlling the stage 30 a, the acceleration α is applied toeach of the adhered cells 96 connected as the aggregated portion 95, andrespective adhered cells 96 are peeled off. As illustrated in FIG. 10,on the adhered cell 96, the same external force F₀ as that on thesuspended cell 90 acts, and in addition to that, an adhesive force F₃generated between the cell and the cell acts in a direction opposite tothe direction of the external force F_(0.) For this reason, a force ofF₀-F₃(=F₄) acts on the adhered cell 96 connected to another adhered cell96 in the aggregated portion 95, and by the force, the adhered cell 96is peeled off from another adhered cell 96 in the aggregated portion 95.An index regarding the acceleration generated with respect to theadhered cell 96 peeled off at this time is set to α′ (α′<α). Forexample, a moving distance D′ of the adhered cell 96 and a speed of theadhered cell 96 from when the stage 30 a is stopped to when the periodof time t elapses can be determined from the obtained entire images, sothat from these values, it is possible to calculate the index α′regarding the acceleration with respect to the peeled-off adhered cell96. By calculating the index α′ regarding the acceleration, the force F₄that acts on the peeled-off adhered cell 96 is calculated. Bycalculating the force F₄ that acts on the peeled-off adhered cell 96,the adhesive force F₃ between the cell and the cell is determined as theadhesive strength between the cell and the cell.

Step S209 corresponds to a process of determining the presence/absenceof the stirring process based on the determined adhesive strengths. Byexecuting the process in step S206, the adhesive strength F₁ between thecell and the surface for adhesion is determined, and by executing theprocess in step S208, the adhesive strength F₃ between the cell and thecell is determined. The CPU 74 reads the second stirring data 79 fromthe memory 73, and determines the presence/absence of the stirringprocess based on the adhesive strength F₁ between the cell and thesurface for adhesion, the adhesive strength F₃ between the cell and thecell and the second stirring data 79. For example, if the adhesivestrength F₁ between the cell and the surface for adhesion becomessmaller than the adhesive strength F₃ between the cell and the cell, theaggregated portion 95 is generated in a process of culturing the cells,so that the CPU 74 sets that the stirring process is performed (presenceof stirring process). On the other hand, if the adhesive strength F₁between the cell and the surface for adhesion becomes larger than theadhesive strength F₃ between the cell and the cell, the aforementionedaggregated portion 95 is not generated in a process of culturing thecells, so that the CPU 74 sets that the stirring process is notperformed (absence of stirring process).

Step S210 corresponds to a process of determining whether or not thereexists the stirring process. When “presence of stirring process” is setin the process in step S209, the CPU 74 sets a result of thedetermination process in the step S210 to Yes, and the process proceedsto step S211. On the other hand, when “absence of stirring process” isset in the process in step S209, the CPU 74 sets a result of thedetermination process in the step S210 to No, and the process proceedsto step S213.

Step S211 corresponds to a process of determining the intensity leveland the number of times of stirring in the stirring process. Note thatthe process in the step S211 is similar to the process in step S104, sothat details thereof will be omitted. Note that in the step S210, theCPU 74 refers to the second stirring data 79 to determine the intensitylevel and the number of times of stirring in the stirring process.

Step S212 corresponds to a process of generating the observationschedule with the performance of stirring process. The process in thestep S212 is similar to the process in step S105, so that detailsthereof will be omitted here.

Step S213 corresponds to a process of generating the observationschedule without the performance of stirring process. The process in thestep S213 is similar to the process in step S106, so that detailsthereof will be omitted here.

Step S214 corresponds to a process of registering the generatedobservation schedule. The process in the step S214 is similar to theprocess in step S107, so that details thereof will be omitted here.Accordingly, even when the type of the cell and the type of the culturevessel 31 to be used are not previously known, by measuring the adhesivestrength between the cell and the cell and the adhesive strength betweenthe cell and the surface for adhesion, the presence/absence of thestirring process, and the intensity level and the number of times ofstirring in the stirring process when performing the stirring process,are automatically determined, and then the observation schedule based onthe determined content is generated. By culturing cells based on thegenerated observation schedule, it is possible to prevent the seedingvariation that occurs in a process of performing the culture, and theoccurrence of density variation of cells proliferated in a process ofperforming the culture, which enables to efficiently culture the cells.

Note that in the present embodiment, the observation schedule isgenerated by using the measurement mode when the type of the cell andthe type of the culture vessel 31 to be used are not known, but, thereare cases in which, for example, even when the type of the cell and theculture vessel 31 used for the culture are known, a combination of theseis not registered in the first stirring data 78, and intended cells arewanted to be cultured after reconfirming whether or not the aggregatedportion 95 is actually generated. In such cases, it is also possible todesign such that the aforementioned measurement mode can be executedafter inputting a type of cell and a culture vessel to be used. Thisenables to deal with a case where, even with a combination of the typeof the cell and the type of the culture vessel with which theobservation schedule with no performance of stirring process isgenerated in the previously registered data, the aggregated portion 95is generated at the time of actually performing the culture, and thelike. In such a case, it is only required that the CPU 74 refers to thesecond stirring data 79 without using the first stirring data todetermine the presence/absence of the stirring process and to determinethe intensity level and the number of times of stirring when performingthe stirring process, thereby generating the observation schedule.

In the present embodiment, by inputting the type of the cell and thetype of the culture vessel 31 to be used, the presence/absence of thestirring process, and the intensity level and the number of times ofstirring in the stirring process when performing the stirring process,are determined. Among the culture vessels 31 used at the time ofactually culturing the cells, there is one whose surface for adhesion issubjected to special surface treatment. In order to allow the use ofsuch culture vessel 31, to the information input through the input unit65 b, a type of surface treatment on the culture vessel 31 may also beadded, in addition to the type of the cell and the type of the culturevessel. In this case, it is also possible to design such that the inputis directly made through the input unit 65 b or the selection can bemade from a list.

In the present embodiment, the adhesive strength (adhesive force)between the cell and the cell and the adhesive strength (adhesive force)between the cell and the surface for adhesion are determined, but, thereis no need to limit to this, and it is only required to detect amagnitude relation of the ease of peeling by giving an external force tothe cells adhered to the inner surface of the culture vessel and anothercultured cell. Further, it is not necessarily required to correctlydetermine the strength and the acceleration after the cell is peeledoff, and it is only required to determine an index by which the adhesivestrength between the cell and the inner surface of the culture vessel orthe adhesive strength between mutual cells can be analogized(estimated). For example, it is also possible that a ratio between themoving distance of the suspended cell and the moving distance of thepeeled-off adhered cell, and a ratio between the moving distance of thesuspended cell and the moving distance of the adhered cell peeled offfrom the aggregated portion, are determined, the ratios are handled asindexes regarding the adhesive strengths, it is determined whether ornot the stirring process is executed, and the intensity level and thenumber of times of stirring in the stirring process are determined whenexecuting the stirring process, based on these ratios, and then theobservation schedule is generated. Further, other than this, it is alsopossible that a ratio between the speed of the suspended cell and thespeed of the peeled-off adhered cell, and a ratio between the speed ofthe suspended cell and the speed of the adhered cell peeled off from theaggregated portion, are determined, it is determined whether or not thestirring process is executed, and the intensity level and the number oftimes of stirring in the stirring process are determined when executingthe stirring process, based on these ratios, and then the observationschedule is generated.

In the present embodiment, the cell to be a target when calculating theadhesive strength between the cell and the surface for adhesion and theadhesive strength between the cell and the cell is set to one cell, but,there is no need to limit to this, and a plurality of cells may also beset as targets. In this case, as the aforementioned adhesive strength,an average value of the adhesive strengths calculated for respectivecells may be determined.

In the present embodiment, the cell culture apparatus that culturescells is adopted as an example, but, there is no need to limit to this,and it is also possible to adopt a program for making the CPU of thecell culture apparatus execute the functions of the image analyzingsection 81, the operating section 82, the schedule management section 83and the timer 84 illustrated in FIG. 5, and the function of the flowcharts illustrated in FIG. 6 and FIG. 7. In this case, when the programis installed in the cell culture apparatus, there is a need to write theoperation data 77, the first stirring data 78, and the second stirringdata 79 into the memory and the database section. Note that theaforementioned program and data are preferably stored in a storagemedium such as a memory card, an optical disk, a magnetic disk or thelike, for example.

The many features and advantages of the embodiments are apparent fromthe detailed specification and, thus, it is intended by the appendedclaims to cover all such features and advantages of the embodiments thatfall within the true spirit and scope thereof. Further, since numerousmodifications and changes will readily occur to those skilled in theart, it is not desired to limit the inventive embodiments to the exactconstruction and operation illustrated and described, and accordinglyall suitable modifications and equivalents may be resorted to, fallingwithin the scope thereof.

1. A stirring method, comprising: measuring, with respect to cells whichadhere to an inner surface of a culture vessel to be cultured, an easeof peeling between the culture vessel and the cell and an ease ofpeeling between the mutual cells; and performing a stirring process onthe cells in the culture vessel by determining a process content of thestirring process with respect to the cells in the culture vessel, basedon the ease of peeling between the culture vessel and the cell and theease of peeling between the mutual cells.
 2. A cell culture method,comprising: seeding the cells in the culture vessel; and stirring thecells cultured in the culture vessel, by using the stirring methodaccording to claim
 1. 3. The cell culture method according to claim 2,wherein the measuring measures the ease of peeling between the culturevessel and the cell and the ease of peeling between the mutual cellscomprises: measuring the ease of peeling between the culture vessel andthe cell by transporting the culture vessel at a predeterminedacceleration and stopping the culture vessel, when the cells are seededin the culture vessel and the cells are fixed to the culture vessel; andmeasuring the ease of peeling between the mutual cells by transportingthe culture vessel at a predetermined acceleration and stopping theculture vessel after the cells are cultured in the culture vessel.
 4. Astirring apparatus, comprising: a stirring unit executing a stirringprocess on cells which adhere to an inner surface of a culture vessel tobe cultured; a stirring process content determining unit determining aprocess content of the stirring process in the stirring unit, inaccordance with a magnitude relation between an ease of peeling betweenthe cell and the inner surface of the culture vessel and an ease ofpeeling between the mutual cells; and a control unit drive-controllingthe stirring unit based on the process content of the stirring processdetermined by the stirring process content determining unit.
 5. Thestirring apparatus according to claim 4, further comprising an inputunit inputting a type of the culture vessel and a type of the cell,wherein the stirring process content determining unit estimates themagnitude relation based on the type of the culture vessel and the typeof the cell input by the input unit, and determines the content of thestirring process in the stirring unit.
 6. The stirring apparatusaccording to claim 5, further comprising a storage unit storing stirringprocess information to which the process content of the stirring processis corresponded, in accordance with the type of the culture vessel andthe type of the cell, wherein the stirring process content determiningunit determines the process content of the stirring process in thestirring unit by refereeing to the stirring process information when thetype of the culture vessel and the type of the cell are input by theinput unit.
 7. The stirring apparatus according to claim 4, furthercomprising: a culture vessel transporting unit giving an acceleration tothe culture vessel; an observing unit observing movements of cells inthe culture vessel; and an analyzing unit determining the magnituderelation by analyzing an observation result of the cells obtained by theobserving unit.
 8. The stirring apparatus according to claim 4, whereinthe magnitude relation is determined as a magnitude relation between anadhesive strength between the cell and the inner surface of the culturevessel and an adhesive strength between the mutual cells.
 9. Thestirring apparatus according to claim 7, wherein the magnitude relationis determined as a magnitude relation between a moving distance when thecell adhered to the culture vessel is peeled off from the culture vesselwhen applying the acceleration and a moving distance of the cell of themutually adhered cells when the mutual cells are peeled off from eachother.
 10. The stirring apparatus according to claim 4, wherein theprocess content of the stirring process is formed of thepresence/absence of the stirring process, the acceleration given to theculture vessel when executing the stirring process and a number of timesof giving the acceleration.
 11. A cell culture apparatus, comprising:the stirring apparatus according to claims 4; and atemperature-controlled room including the stirring apparatus in aninside thereof and maintaining an environment to a state of culturingthe cells.
 12. A stirring apparatus, comprising: a stirring unitexecuting a stirring process with respect to cells which adhere to aninner surface of a culture vessel to be cultured; an input unitinputting a type of the culture vessel and a type of the cell; a storageunit storing a process content of the stirring process determined basedon previously determined ease of peeling between the culture vessel andthe cell and ease of peeling between the mutual cells from a combinationof the type of the culture vessel and the type of the cell input by theinput unit; and a control unit drive-controlling the stirring unit basedon the process content of the stirring process read from the storageunit based on the type of the culture vessel and the type of the cell.13. A cell culture apparatus, comprising: the stirring apparatusaccording to claim 12; and a temperature-controlled room including thestirring apparatus in an inside thereof and maintaining an environmentto a state of culturing the cells.